Spatiotemporal dynamics of macrophage heterogeneity and a potential function of Trem2hi macrophages in infarcted hearts

Heart failure (HF) is a frequent consequence of myocardial infarction (MI). Identification of the precise, time-dependent composition of inflammatory cells may provide clues for the establishment of new biomarkers and therapeutic approaches targeting post-MI HF. Here, we investigate the spatiotemporal dynamics of MI-associated immune cells in a mouse model of MI using spatial transcriptomics and single-cell RNA-sequencing (scRNA-seq). We identify twelve major immune cell populations; their proportions dynamically change after MI. Macrophages are the most abundant population at all-time points (>60%), except for day 1 post-MI. Trajectory inference analysis shows upregulation of Trem2 expression in macrophages during the late phase post-MI. In vivo injection of soluble Trem2 leads to significant functional and structural improvements in infarcted hearts. Our data contribute to a better understanding of MI-driven immune responses and further investigation to determine the regulatory factors of the Trem2 signaling pathway will aid the development of novel therapeutic strategies for post-MI HF.

The time course of immunostaining of Tmem2 and that of western do not match well. Does this reflect the changes in TMEM2+ macrophage number rather than expression levels of TMEM2 in macrophages? Also, on single-cell data TMEM2+ macrophage fraction may stay less than 50% even on days 3 and 5, though the FACS showed otherwise. Did the sections shown contain only these TMEM2+ macrophage populations?
While the data shown in Fig. 6 suggest an overall beneficial effect of sTREM2, it remains unknown as to how TREM2 improved the consequence of MI. What are the cells that are affected by sTREM2? M1/M2 dichotomy was proposed based on the findings of cultured macrophages, and it is now clear that the dichotomy cannot be used to classify macrophages in tissues in vivo (Circulation Research. 2016;119:414-417). The authors label tissue-resident macrophages as M2 and suggest that monocyte-derived macrophages differentiate into M2 after MI. This is problematic for several reasons. First, the marker gene expression pattern of cardiac tissue resident macrophages may not fit the M2 markers. For instance, the authors showed a typical M2 marker, Arg1, was high in TREM2+ macrophages, which may be a transient population that appears late (i.e., 3-7 days) after MI. They are likely to be monocyte-derived cells as opposed to the fetal liverderived tissue resident macrophages in healthy heart. As such, it is unclear why tissue resident macrophages in the steady-state and the macrophages appear late and resembles tissue-resident macrophages should be considered M2. Second, the differentiation trajectories inferred from scRNA data do not necessarily show the actual differentiation course of cells. Because the cells are clustered based on the similarities in transcriptomes and projected on UMAP, and the trajectories are mapped on it, the results may look like a sequential differentiation from Ly6Chi to resident macrophages. However, actual differentiation processes need further biological and computational analyses. For instance, the late macrophage populations may predominate on days 5-7, and they may have transcriptomes similar to those of the resident macrophages. However, they might disappear after this time point and may never differentiate into the resident macrophages. It is also possible that monocytes recruited on day 1 become M1, but those recruited on day 7 directly become TREM2+ cells. As such, the results shown in this study are not sufficient to support the continuum model of differentiation from M1 to M2. Minor p. 11 l.244 Fig.5b should be 5c.

Reviewer #2 (Remarks to the Author):
The authors investigated the spatiotemporal dynamics of MI-associated immune cells in a murine model of MI using single-cell RNA-sequencing and spatial transcriptomics. They describe the heterogeneity, compositional changes, and approximate spatial localization of immune cell populations in their model. They identify upregulation of Trem2 expression in macrophages and show that in vivo injection of soluble Trem2 resulted in functional and structural improvement in infarcted hearts. These results are of significance to the field.
In general, the work supports the conclusions and claims and there are no flaws in the data analysis, interpretation and conclusions that prohibit publication or require revision. The authors used standard scRNA-seq computational analysis pipelines and methodology meets the expected standards in this field. There is sufficient detail provided in the methods for the work to be reproduced.

I have a few minor questions/suggestions for the authors:
Line 117: "Three macrophage sub-clusters (clusters 1, 3, and 13) were predominant in the steady-state (77.9% of the total monocyte/macrophages), suggesting that they are tissue-resident macrophages (termed Res-Mφ clusters, Fig. 3b)." Not sure that the predominance of these subclusters proves that they are tissue-resident macrophages. Are there any markers or techniques (eg macrophage-marker IV negative labeling) that would prove this? Macrophage clustering seen in ST-seq is interesting, but of course it should be stated that this is not technically single-cell, so the authors are using cell type signatures as an approximate approach to identify cell types. Nothing to do about this, just that this caveat should be stated in the manuscript.
Line 179: "When Ly6c2hi monocytes were set as the root of the trajectory" What is the justification for setting Ly6c hi monocytes as the root? Line 192: "Importantly, when we checked the expression of candidate genes in injured heart tissues by ST-seq, marker genes for each subset were enriched in the infarcted area of the heart in a time-dependent manner, while Res-Mφ1 cells were dispersed in the heart for all time points after MI (Fig. 3e, Supplementary Fig. 5). Supp Fig 5 uses four putative marker genes for res-Mac1 "subset", but the expression for each gene is rather sparse and does not prove that these genes are expressed specifically by macrophages. Imputed trajectory analyses (especially using the original Monocle algorithm) are not without caveats (eg artificially forcing trajectories), which should be stated somewhere in the paper.

Do the authors envision that injection of soluble Trem2 would have translational relevance? What cells other than macrophages might respond to Trem2?
Reviewer #3 (Remarks to the Author): Single-cell transcriptomics unveils the spatiotemporal dynamics of macrophage heterogeneity and a potential role of Trem2hi macrophages in infarcted hearts In this manuscript, the authors have applied the single-cell RNA sequencing and spatial transcriptome sequencing to identify different populations of immune cells that operate at different time points and with different dynamics following MI. In particular, chronic stage such as 28 days. Authors inject sTrem2 right after MI and it showed protective effect, what is mechanism behind? Is it due to transition of macrophages phenotype or anti-inflammatory response to entire heart? Authors need more evidence to clarify the cardioprotective effect of Trem2.
13. Tremhi macrophages exhibit more M2 phenotypes with increased Arg1 level, however this result is not sufficient to conclude Tremhi macrophages are antiinflammatory macrophages. Facs analysis of Tremhi macrophages are required for better understanding of the effect of Trem on macrophage polarization.
14. Despite M2 macrophages present anti-inflammatory characters, they often show also pro-fibrotic features. Authors didn't show any results related to fibrosis or fibroblasts. Additional data about the effect of sTrem2 on fibrosis are required. Fig.6C, it would be interesting to show the results compared to the control group of mice without LAD ligation. Moreover, it seems that the gelatin hydrogel without Trem2 significantly improves the LVEF and FS compared to the PBS treatment. Authors need clarify that in discussion.

Minor Remarks
The authors should describe the number of biological replicates for each experiment. In the introduction is mentioned the nomenclature M1 and M2 that is obsolete. The in vivo environment is more complex, as demonstrated by this paper, so this nomenclature can be limited only to an in vitro environment. I would suggest to the authors to specify this or to edit it (PMID: 27458196). Since Trem2 is the crucial molecule discussed in this manuscript, this should be mentioned and briefly described also in the introductory part, not only in the discussions. The results of Fig.1 and Fig.2 should be better discussed. For Fig.2 the conclusion is completely missed. Page 11, line 244, there is a mistake in the mentioned Figure. It should be 5c and not 5b. Fig.5d-f requires an extensive explanation. In material and methods, paragraph co-localization assay, the reference of the secondary antibodies is missing.

Reviewer #1
In this manuscript, authors have analyzed CD45 + cells after MI using scRNA-seq and Visium.
Previous studies have shown that macrophage populations and functions are temporally changed after MI. Therefore, it is important to further characterize the functional and phenotypic diversities in cardiac macrophages after MI. The authors show the temporal changes in macrophage subsets and provide data suggesting TREM2 may have a cardioprotective function after MI. Comment 1. In Figure 2, the authors used the Visium system to analyze the spatial transcriptome of the myocardium. The authors described, "Most of the mouse heart cell were cardiomyocytes…". This is misleading and contradicts the previous careful studies of the cardiac cell populations (AJP 293:H1883-H1891, 2007Circ Res 118:400-409, 2016).
Nonmyocytes, including fibroblasts and macrophages, are known to be scattered in the myocardium (Cell 183:94-109.e123, 2020.). Because the resolution of methods is noted as 1-10 cells, it is likely the inclusion of RNA from cardiomyocytes, which would cluster myocytecontaining spots as a cluster expressing myocyte markers, resulting in an overestimation of the number of myocytes. Authors should consider revising the part to more precisely state what can be concluded from the results.

Response:
We agree with the reviewer's comment on the ST-seq data. As the reviewer suggested, we have reanalyzed the ST-seq data using SPOTlight, a nonnegative matrix factorization (NMF)-based spatial deconvolution framework (PMID: 33544846), to infer the cell-type composition of each spot. SPOTlight identified cell-type-specific gene expression signatures from scRNA-seq data, which were subsequently used to deconvolute ST-seq spots.
Based on the SPOTlight analysis, we have revised Figure 2 and added Supplementary Figures 2-5 and 11. Because our scRNA-seq data included only cytometry-sorted CD45 + leukocytes, statements regarding the cardiomyocyte have been removed from the manuscript. In addition, we have replaced the ST-seq data of the day 5 post-MI sample, which was noted as an inappropriate image by reviewer #3.
The corrections in the Result section of the revised manuscript are as follows. "We then applied SPOTlight (PMID: 33544846), a nonnegative matrix factorization (NMF)-based spatial deconvolution framework, to infer the cell-type composition of each spot (Fig. 2b). Notably, neutrophils highly infiltrated into the infarcted area on day 1 (41.2% on average) but their numbers were then decreased rapidly (18.7, 10.6, and 9.5% on days 3, 5, and 7, respectively), which was consistent with the scRNA-seq data ( Fig. 2b and 2c). On the contrary, macrophages were dispersed across the whole heart rather than clustered in the infarcted area on day 1 (Fig.   2b). However, from day 3, macrophages infiltrated into the infarcted area, and their abundance peaked at late MI (days 5 and 7; 18.8% on average) (Fig. 2c). Monocytes and fibroblasts also infiltrated the infarcted area of the heart in a time-dependent manner (Supplementary Fig. 1-5).
Other immune cell populations, such as B-cell and T-cell populations, were always dispersed across the entire mouse heart (not clustered in the infarcted area), with a very low abundance . Together with the scRNA-seq showing immune cell temporal dynamics after MI, ST-seq analysis with deconvolution algorithm further provided their spatial heterogeneity and spatiotemporal dynamics; our ST-seq data clearly showed that monocytes and neutrophils infiltrated into the infarcted area at early MI while macrophages and fibroblasts acted oppositely." (page 7-8, line 119-135) We have also added a statement on the caveat for the ST-seq in the Discussion section as follows. "Although ST-seq used in this study does not provide spatial expression profiles at true single-cell resolution (1-10 cell resolution on average per spot), ST-seq analysis with deconvolution algorithm revealed that the proportions of monocytes, neutrophils, and macrophages increased or decreased in a time-dependent manner, which was consistent with previous reports." (page 20, line 422-426) Comment 2. The time course of immunostaining of Trem2 and that of western do not match well. Does this reflect the changes in TREM2 + macrophage number rather than expression levels of TREM2 in macrophages? Also, on single-cell data TMEM2 + macrophage fraction may stay less than 50% even on days 3 and 5, though the FACS showed otherwise. Did the sections shown contain only these TMEM2 + macrophage populations?

Response:
We appreciate this valuable comment. In Figure 5b, we intended to confirm the expression dynamics of Trem2 over time after MI, in which we used the anti-Trem2 antibody targeting common C-terminals contained in both full-length Trem2 and its soluble form (sTrem2). The results showed that the expression of full-length Trem2 peaked on day 5 post-MI and decreased thereafter, whereas that of sTrem2 appeared on day 3 and peaked on day 7.
Thus, the expression of total Trem2 (full-length Trem2 and sTrem2) steadily increased, peaking 5 days post-MI and decreasing thereafter. We believe these results are compatible with those obtained in Trem2 IHC analysis and co-localization assay. To clarify this, we have changed "upper bands from Trem2" to "full-length Trem2" and added the quantitative analysis data for comparison of relative Trem2 levels in the revised Figure 5b and 5c.
<Revised Figure 5a-5c> The discrepancy in Trem2 hi macrophage populations between scRNA-seq and FACS data seems to be due to the difference between the two techniques. In scRNA-seq, as the reviewer mentioned in comment 4, CD45 + leukocytes isolated from infarct and peri-infarct areas were clustered based on the similarities in transcriptomes, not Trem2 expression. Thus, the gene signature-based clustering can cause an underestimation of the number of Trem2-expressing macrophages. Indeed, some macrophages expressing Trem2 were grouped into other macrophage subsets, such as Transient-Mφ2, Transient-Mφ3, and Late-Mφ1 (Revised Supplementary Figure 7). In contrast, in FACS analysis, single-cell suspensions were sorted into CD45 + F4/80 + macrophages to collect Trem2 hi macrophage subsets specifically. Although a relatively high Trem2 hi macrophage proportion was observed in FACS analysis compared to scRNA-Seq, the trends (dominance in late phase post-MI) were consistent for both methods. Comment 3. While the data shown in Fig. 6 suggest an overall beneficial effect of sTREM2, it remains unknown as to how TREM2 improved the consequence of MI. What are the cells that are affected by sTREM2?

Response:
We appreciate the reviewer's valuable comment. Regarding this issue, we first analyzed the expression level of Arginase 1 (Arg1) to characterize the polarizing status of the Ccr2 hi and Trem2 hi macrophage subsets. Significantly higher expression levels of Arg1 were observed in Trem2 hi macrophages than that in Ccr2 hi macrophages (revised Fig. 5h). Next, we quantified inflammatory and anti-inflammatory mRNA expression levels in Ccr2 hi and Trem2 hi macrophages (revised Fig. 5i and 5j). Ccr2 hi macrophages exhibited higher expression levels of pro-inflammatory cytokines, such as IL-1β and IL-6, whereas Trem2 hi macrophages expressed higher levels of anti-inflammatory cytokines, such as IL-4, IL-10, and Alox15. In addition, Trem2 hi macrophages augmented anti-inflammatory chemokines, such as Ccl1 and Cxcr1, as well as anti-inflammatory signature genes such as TGF-β. By contrast, Ccr2 hi macrophages expressed higher levels of pro-inflammatory chemokines, such as Ccl2, Cxcr3, and Cxcr7. These results indicate the anti-inflammatory nature of Trem2 hi macrophages in contrast with early-stage Ccr2 hi macrophages. Interestingly, Trem2 hi macrophages exhibited a higher expression level of osteopontin (Spp1), which is related to pro-fibrotic potential in regulating post-MI LV remodeling (PMID: 11375279, 31411676). These results indicate that Trem2 hi macrophages may have anti-inflammatory macrophage characteristics.
<Revised figure 5i and 5j> Although we found the beneficial anti-remodeling effect of sTrem2 post-MI, it will be a difficult task to investigate the underlying mechanisms, such as what type of cells are affected by sTrem2 and how sTrem2 induces intracellular signaling. Late-stage Trem2 macrophages may secrete sTrem2 and can affect the macrophage itself via autocrine regulation. To investigate how sTrem2 affects the macrophage, we tested the polarizing effect of sTrem2 on thioglycolate-elicited peritoneal macrophages from C57BL/6 mice. When peritoneal macrophages were activated toward a pro-inflammatory phenotype by lipopolysaccharide (LPS, 1 μg/mL) and interferon-γ (IFN-γ, 4 ng/mL) treatments, IL-12B and iNOS (proinflammatory markers) were upregulated while Arg1 (anti-inflammatory marker) was downregulated. On the contrary, sTrem2 (200 ng/ml) treatment after LPS and IFN-γ treatments decreased or increased the expression levels of pro-inflammatory and anti-inflammatory markers, respectively (see the figure below). This finding suggests that sTrem2 affects macrophage polarization, but since further detailed research should be performed to draw a firm conclusion, we did not include these in vitro data in the revised manuscript. We agree with the reviewer's concern about the Trem2 function in MI, but the exploration of more possibilities is beyond our capacity at this time and will be carried out in future studies. This statement has been added to the Discussion section of the revised manuscript as follows: "Further studies are, however, needed to systematically evaluate the Trem2 hi macrophage origin, the cells affected by sTrem2, and the related intracellular signaling pathways, to enable a better understanding of its regenerative effect in infarcted hearts, before conducting human clinical trials." (page 19, line 397-400) < in vitro polarization in peritoneal macrophages > Comment 4. M1/M2 dichotomy was proposed based on the findings of cultured macrophages, and it is now clear that the dichotomy cannot be used to classify macrophages in tissues in vivo (Circulation Research. 2016;119:414-417). The authors label tissue-resident macrophages as M2 and suggest that monocyte-derived macrophages differentiate into M2 after MI. This is problematic for several reasons. First, the marker gene expression pattern of cardiac tissue resident macrophages may not fit the M2 markers. For instance, the authors showed a typical M2 marker, Arg1, was high in TREM2 + macrophages, which may be a transient population that appears late (i.e., 3-7 days) after MI. They are likely to be monocyte-derived cells as opposed to the fetal liver-derived tissue resident macrophages in healthy heart. As such, it is unclear why tissue resident macrophages in the steady-state and the macrophages appear late and resembles tissue-resident macrophages should be considered M2. Second, the differentiation trajectories inferred from scRNA data do not necessarily show the actual differentiation course of cells. Because the cells are clustered based on the similarities in transcriptomes and projected on UMAP, and the trajectories are mapped on it, the results may look like a sequential differentiation from Ly6Chi to resident macrophages. However, actual differentiation processes need further biological and computational analyses. For instance, the late macrophage populations may predominate on days 5-7, and they may have transcriptomes similar to those of the resident macrophages. However, they might disappear after this time point and may never differentiate into the resident macrophages. It is also possible that monocytes recruited on day 1 become M1, but those recruited on day 7 directly become TREM2+ cells. As such, the results shown in this study are not sufficient to support the continuum model of differentiation from M1 to M2.

Response:
We agree with the reviewer's concern. As suggested, the M1/M2 dichotomy is no longer used in the revised manuscript and related sentences were consistently changed in the Introduction as follows: "However, this dichotomous classification was obsolete, as in vivo environment of macrophages are more complex (PMID: 27458196). Therefore, a more precise evaluation is needed for accurate characterization of the dynamics of macrophage heterogeneity during the acute period of MI." (page 4, line 61-64) The following was added to the Discussion: "third, the single-cell trajectory analysis supports a sequential differentiation from Ly6c2 hi monocytes to Late-Mφ rather than the obsolete dichotomous M1-M2 paradigm" (page 17, line 335-337) Filip K. Swirski, the author of the paper the reviewer mentioned and one of the authors of this manuscript, has read and approved this manuscript.
In addition, the trajectory analysis was re-performed by excluding the tissue-resident macrophages (see the figure below), and caveats for inferred trajectories have been added in the Discussion as follows. "Although our inferred trajectories harbored a limitation that Ly6c2 hi monocytes were artificially designated into root state with simple tree-structure models (PMID:

Response:
We have corrected this mistake in the revised manuscript. Thank you.

Reviewer #2
The authors investigated the spatiotemporal dynamics of MI-associated immune cells in a murine model of MI using single-cell RNA-sequencing and spatial transcriptomics. They describe the heterogeneity, compositional changes, and approximate spatial localization of immune cell populations in their model. They identify upregulation of Trem2 expression in macrophages and show that in vivo injection of soluble Trem2 resulted in functional and structural improvement in infarcted hearts. These results are of significance to the field. In general, the work supports the conclusions and claims and there are no flaws in the data analysis, interpretation and conclusions that prohibit publication or require revision. The authors used standard scRNA-seq computational analysis pipelines and methodology meets the expected standards in this field. There is sufficient detail provided in the methods for the work to be reproduced.

Response:
We thank the reviewer for recognizing this work as worthy of publication. Comment 1. (Minor points) Line 117: "Three macrophage sub-clusters (clusters 1, 3, and 13) were predominant in the steady-state (77.9% of the total monocyte/macrophages), suggesting that they are tissue-resident macrophages (termed Res-Mφ clusters, Fig. 3b)." Not sure that the predominance of these subclusters proves that they are tissue-resident macrophages. Are there any markers or techniques (eg macrophage-marker IV negative labeling) that would prove this?
Macrophage clustering seen in ST-seq is interesting, but of course it should be stated that this is not technically single-cell, so the authors are using cell type signatures as an approximate approach to identify cell types. Nothing to do about this, just that this caveat should be stated in the manuscript.

Response:
We appreciate this comment. In a recent work by Dick et al. (PMID: 30538339), scRNA-seq analysis of mouse heart tissue at steady state identified four distinct macrophage clusters (TIMD4, MHC-II, ISG, and CCR2 Mφ subsets) as tissue-resident cardiac macrophages.
The fate-mapping analysis further showed that CCR2 -TIMD4 + Mφ population was maintained independently of monocytes: CCR2 -TIMD4 -MHCII hi Mφ were partially replaced by monocytes and CCR2 + MHCII hi Mφ were fully replaced by monocytes over time. In our study, two specific findings seemed to support our interpretation: 1) Three macrophage sub-clusters (clusters 1, 3, and 13) were predominant in the steady-state, not post-MI state; 2) cluster 3 macrophages (Res-Mφ1) showed elevated expression of TIMD4 Mφ-related genes (Lyve1, Response: Mouse Ly6c hi monocytes share several properties with human CD16 -CD14 + monocytes and they are inflammatory. In addition, it is well known that when the Ly6c hi monocytes enter the circulation, contribute to excessive monocytosis, preferentially accumulate around lesions, and differentiate into macrophages after MI (PMID: 23307733, 17200719, and 22144566). Therefore, we set the Ly6c2 hi monocytes as the root state of the trajectory analysis. This statement has been added to the Results section of the revised manuscript as follows. "Ly6c2 hi monocytes share several properties with human CD16 -CD14 + monocytes, and it is well known that they enter the circulation, contribute to excessive monocytosis, preferentially accumulate in lesions, and differentiate into macrophages after MI." uses four putative marker genes for res-Mac1 "subset", but the expression for each gene is rather sparse and does not prove that these genes are expressed specifically by macrophages.
Imputed trajectory analyses (especially using the original Monocle algorithm) are not without caveats (eg artificially forcing trajectories), which should be stated somewhere in the paper.

Response:
We appreciate this comment. In this revision, we have reanalyzed the ST-seq data using SPOTlight, a nonnegative matrix factorization (NMF)-based spatial deconvolution framework (PMID: 33544846), to infer the cell-type composition of each spot. SPOTlight we tested the polarizing effect of sTrem2 on thioglycolate-elicited peritoneal macrophages from C57BL/6 mice. When peritoneal macrophages were activated toward a pro-inflammatory phenotype by lipopolysaccharide (LPS, 1 μg/mL) and interferon-γ (IFN-γ, 4 ng/mL) treatments, IL-12B and iNOS (pro-inflammatory markers) were upregulated while Arg1 (anti-inflammatory marker) was downregulated. On the contrary, sTrem2 (200 ng/ml) treatment after LPS and IFNγ treatments decreased or increased the expression levels of pro-inflammatory and antiinflammatory markers, respectively (see the figure below). This finding suggests that sTrem2 affects macrophage polarization, but since further detailed research should be performed to draw a firm conclusion, we did not include these in vitro data in the revised manuscript. We agree with the reviewer's concern about the Trem2 function in MI, but the exploration of more possibilities is beyond our capacity at this time and will be carried out in future studies. This statement has been added to the Discussion section of the revised manuscript as follows: "Further studies are, however, needed to systematically evaluate the Trem2 hi macrophage origin, the cells affected by sTrem2, and the related intracellular signaling pathways, to enable a better understanding of its regenerative effect in infarcted hearts, before conducting human clinical trials." (page 19, line 397-400) < in vitro polarization in peritoneal macrophages >

Reviewer #3
Single-cell transcriptomics unveils the spatiotemporal dynamics of macrophage heterogeneity and a potential role of Trem2 hi macrophages in infarcted hearts. In this manuscript, the authors have applied the single-cell RNA sequencing and spatial transcriptome sequencing to identify different populations of immune cells that operate at different time points and with different dynamics following MI. In particular, macrophages have emerged as the most dominant and enriched population able to release Trem2 in the later stages post-MI. By in vivo injection of soluble Trem2, the authors show functional and structural improvements of the infarcted heart. This paper provides an excellent overview of the interplay of different types and subtypes of immune cells following MI by using innovative techniques. Overall, the manuscript is fluid to read and very informative. Nonetheless, there are a few concerns that should be addressed.

Response:
We thank the reviewer for recognizing this work as worthy of publication. Comment 1. Fig.1d, authors present proportion of macrophages during the 7 days following MI. However, expansion of infarct size associated with MI time frame has not been considered here. Therefore, addition of table with information of infarct size, cell population (cell counts and percentage).

Response:
As the reviewer suggested, we have added the additional table containing infarct size and cell population information to the revised manuscript as Supplementary Table 2.
Comment 2. Fig.2a and 2b, representative image of day 5 post-MI sample is not appropriate. Based on the SPOTlight analysis, the related statements have also been changed as follows: "We then applied SPOTlight (PMID: 33544846), a nonnegative matrix factorization (NMF)based spatial deconvolution framework, to infer the cell-type composition of each spot (Fig.   2b). Notably, neutrophils highly infiltrated into the infarcted area on day 1 (41.2% on average) but their numbers were then decreased rapidly (18.7, 10.6, and 9.5% on days 3, 5, and 7, respectively), which was consistent with the scRNA-seq data ( Fig. 2b and 2c). On the contrary, macrophages were dispersed across the whole heart rather than clustered in the infarcted area on day 1 (Fig. 2b). However, from day 3, macrophages infiltrated into the infarcted area, and their abundance peaked at late MI (days 5 and 7; 18.8% on average) (Fig. 2c). Monocytes and fibroblasts also infiltrated the infarcted area of the heart in a time-dependent manner (Supplementary Fig. 1-5). Other immune cell populations, such as B-cell and T-cell populations, were always dispersed across the entire mouse heart (not clustered in the infarcted area), with a very low abundance ( Supplementary Fig. 2-5). Together with the scRNA-seq showing immune cell temporal dynamics after MI, ST-seq analysis with deconvolution algorithm further provided their spatial heterogeneity and spatiotemporal dynamics; our ST-seq data clearly showed that monocytes and neutrophils infiltrated into the infarcted area at early MI while macrophages and fibroblasts acted oppositely." (page 7-8, line 119-135) Comment 4. In Fig.3B, the authors show a higher presence of resident macrophages at a steady-state, a decrease after MI and restored at days 5-7. Authors need to describe whether the resident macrophages are restored due to proliferation of the remaining resident cells or replacement.

Response: We appreciate this comment. In a recent work by Dick et al. (PMID: 30538339),
scRNA-seq analysis of mouse heart tissue at steady state identified four distinct macrophage clusters (TIMD4, MHC-II, ISG, and CCR2 Mφ subsets) as tissue-resident cardiac macrophages.
The fate-mapping analysis further showed that CCR2 -TIMD4 + Mφ were maintained independently of monocytes: CCR2 -TIMD4 -MHCII hi Mφ were partially replaced by monocytes and CCR2 + MHCII hi Mφ were fully replaced by monocytes over time. Considering that the differentially expressed genes of TIMD4 Mφ (Lyve1, F13a1, Cbr2, Folr2, and Timd4) and MHC-II M0φ (H2-Eb1, H2-Aa, H2-Ab1) resembled those of clusters 3 (Res-Mφ1) and 1 (Res-Mφ2), respectively, Res-Mφ1 might be restored due to proliferation of the remaining resident cells, while Res-Mφ2 might be replaced. To distinguish the monocyte-and embryoderived resident macrophages more clearly, we have consistently changed the term from tissueresident macrophages (Res-Mφ clusters) to steady-state macrophages (SS-Mφ clusters) in the revised manuscript. These descriptions have been added to the Discussion as follows. "Notably, SS-Mφ populations decreased after MI and were restored on days 5-7. Considering the previous study, which reported that resident cardiac CCR2 -TIMD4 + Mφ (SS-Mφ1 in this study) were maintained independently of monocytes, while CCR2 +/-TIMD4 -MHCII hi Mφ (SS-Mφ2 in this study) were fully or partially replaced by monocyte over time (PMID: 30538339), SS-Mφ1 might be restored due to proliferation of the remaining resident cells, while SS-Mφ2 might be replaced. Further in-depth analysis using fate-mapping will provide their conclusive origin." (page 18, line 357-363). Fig.5f, expression of CCR2 and Trem2 are inversely correlated, dose CCR2 positive cells are gradually transit to Trem2 macrophages? Or is Trem2 are fresh population at later stage of MI? Moreover, the late macrophages increase at days 5 and 7, don't express CCR but they are not mentioned as resident macrophages. What is their origin?

Comment 5. In
Response: We agree with the reviewer's concern regarding the origin of Trem2 hi macrophages.
It is well-known that resident cardiac macrophages are primarily established prenatally and arise from embryonic yolk-sac progenitors. As indicated in the response to comment 4, tissueresident macrophages are characterized by unique gene signatures such as Lyve1, F13a1, Cbr2, Folr2, and Timd4. From this point of view, Trem2 hi macrophages did not express the tissueresident macrophage signatures, and their Ccr2 expression was inversely correlated with Ly6c2 hi monocytes. Furthermore, our inferred trajectory of monocytes/macrophages supports a sequential differentiation from Ly6c2 hi monocytes to Trem2 hi macrophages (revised figure   3c). Therefore, we interpreted that the Ccr2-positive cells gradually differentiate into Trem2 hi macrophages. To this end, experiments, such as fate-mapping, will be helpful, but this was beyond our capabilities. We have added this limitation to the Discussion as follows: "Further studies are, however, needed to systematically evaluate the Trem2 hi macrophage origin, the cells affected by sTrem2, and the related intracellular signaling pathways, to enable a better understanding of its regenerative effect in infarcted hearts, before conducting human clinical trials" (page 19, line 397-400) Comment 6. Authors provide IHC image and co-staining of Trem2 and cardiac macrophages, however, whether macrophages are only cell source of Trem2 is not clear. Measurement of Trem2 expression in all cardiac cell fraction is recommended.
Response: As suggested, we performed Trem2 and cardiac macrophage marker (CD68) costaining in all cardiac cell fractions on day 5 post-MI. The CD68 + macrophage proportions increased markedly in the infarcted area and the Trem2 signals overlapped with those of CD68 + cells, suggesting that the macrophages are the major cell source of Trem2. We have added these results to the revised manuscript (revised Figure 5c).

<Revised figure 5c>
Comment 7. Please add the quantification of WB in Fig.5b. Additionally, it is required a better description of the full length and soluble form of Trem2.

Response:
As the reviewer suggested, we have added the quantification data for the comparison of relative Trem2 levels and changed "upper bands from Trem2" to "full-length Trem2" in the revised Figure 5b. The description of total Trem2 expression has also been added to the Results section as follows: "The expression of total Trem2 (full-length Trem2 and sTrem2) peaked on day 5 (Fig. 5b)." (page 13, line 269-270).

<Revised figure 5b>
Comment 8. Authors showed soluble fraction of Trem2 increased in later stage of MI. What about in secretome or plasma, is Trem2 commonly detected in animal with MI or heart failure patient?
Response: To answer this, we have compared the sTrem2 levels in the plasma of the MI mouse model (see the figure below). sTrem2 levels increased gradually until 5 days post-MI and then gradually decreased. This trend was largely compatible with that of Trem2-expressing macrophages in the infarcted heart tissue. However, this result did not show statistical significance, so further studies are needed to verify the potential of sTrem2 as a biomarker of MI progression. We did not include this result in the revised manuscript because of the word limitation, and also because this is not the main point of this study. Fig.5c, it would be interesting to integrate the staining for cardiomyocytes.

Comment 9. In
Considering the starting demonstrations in which late macrophages co-localize with the damaged area (Fig.3E), a staining to highlight the infarcted area should be performed in combination with CD68 and Trem2.

Response:
As the reviewer suggested, we performed Trem2 and cardiomyocyte marker (myoglobin) co-staining in the infarcted heart on day 5 post-MI. However, as shown in the revised Figure 5c, Trem2 expression was mostly observed in the macrophages and there was no significant Trem2 expression in the cardiomyocytes (see the figure below). Therefore, the Trem2 and CD68 co-staining images have been added to the revised Figure 5c to highlight the infarcted area.
Comment 10. Fig.6 should be integrated with additional information such as ratio LV mass/BW and a table with morphometric and echocardiographic characteristics of the mice (mortality rate, infarct size, body weight, IVSd, IVSs, LVIDd, LVIDs; etc.). Table would be great addition.
Response: As the reviewer suggested, we have added the survival rate of each group to the revised Figure 6 and the echocardiography parameters of the mice to the revised Supplementary Table 6. However, we were unable to provide the LV mass/BW ratio because we did not measure LV mass or BW. The related description has also been added to the Results section as follows. "The infarct size was also significantly smaller in sTrem2-GH-treated MI mice (26.0%) than that in PBS-(48.4%) and GH-treated (38.0%) mice (Fig. 6e) and an increased survival rate was observed in the mice treated with sTrem2-GH compared with other groups (Fig. 6f) Response: Thank you for this constructive comment. As the reviewer suggested, we have analyzed the long-term changes of sTrem2 expression in the infarcted heart tissues. Western blotting showed that the expression of sTrem2 was significantly decreased in the chronic stage compared with that in the late stage (see the figure below). It is noteworthy that these initial changes in Trem2 expression observed in the infarcted areas for 7 days are closely related to the inflammation-modulating role and anti-remodeling effect in vivo.
Although we found the beneficial anti-remodeling effect of sTrem2 post-MI, it will be a difficult task to investigate the underlying mechanisms, such as what type of cells are affected by sTrem2 and how sTrem2 induces intracellular signaling. Late-stage Trem2 macrophages may secrete sTrem2 and can affect the macrophage itself via autocrine regulation. To investigate how sTrem2 affects the macrophage, we tested the polarizing effect of sTrem2 on thioglycolate-elicited peritoneal macrophages from C57BL/6 mice. When peritoneal macrophages were activated toward a pro-inflammatory phenotype by lipopolysaccharide (LPS, 1 μg/mL) and interferon-γ (IFN-γ, 4 ng/mL) treatments, IL-12B and iNOS (proinflammatory markers) were upregulated while Arg1 (anti-inflammatory marker) was downregulated. On the contrary, sTrem2 (200 ng/ml) treatment after LPS and IFN-γ treatments decreased or increased the expression levels of pro-inflammatory and anti-inflammatory markers, respectively (see the figure below). This finding suggests that sTrem2 affects macrophage polarization, but since further detailed research should be performed to draw a firm conclusion, we did not include these in vitro data in the revised manuscript. We agree with the reviewer's concern about the Trem2 function in MI, but the exploration of more possibilities is beyond our capacity at this time and will be carried out in future studies. This statement has been added to the Discussion section of the revised manuscript as follows: "Further studies are, however, needed to systematically evaluate the Trem2 hi macrophage origin, the cells affected by sTrem2, and the related intracellular signaling pathways, to enable a better understanding of its regenerative effect in infarcted hearts, before conducting human clinical trials." (page 19, line 397-400) Comment 13. Trem hi macrophages exhibit more M2 phenotypes with increased Arg1 level, however this result is not sufficient to conclude Trem hi macrophages are anti-inflammatory macrophages. Facs analysis of Trem hi macrophages are required for better understanding of the effect of Trem on macrophage polarization.

Response:
We appreciate this comment. As the reviewer suggested, we have further analyzed the polarizing status of macrophage subsets by quantifying inflammatory and antiinflammatory mRNA expression levels in the Ccr2 hi and Trem2 hi macrophages (revised Fig. 5i and 5j). Ccr2 hi macrophages exhibited higher expression levels of pro-inflammatory cytokines (IL-1β and IL-6) and chemokines (Ccl2, Cxcr3, and Cxcr7), whereas Trem2 hi macrophages expressed higher levels of anti-inflammatory cytokines (IL-4, IL-10, and Alox15), chemokines (Ccl1 and Cxcr1), as well as the anti-inflammatory signature gene, TGF-β.
<Revised figure 5i and 5j> Consistent with our conclusions, these data suggest that Trem2 hi macrophages possess antiinflammatory characteristics. Related statements have been added and to the Results section as follows: "Next, we quantified inflammatory and anti-inflammatory mRNA expression levels in the Ccr2 hi and Trem2 hi macrophages. Ccr2 hi macrophages exhibited higher expression levels of pro-inflammatory cytokines (IL-1β and IL-6) and chemokines (Ccl2, Cxcr3, and Cxcr7), whereas Trem2 hi macrophages expressed higher levels of anti-inflammatory cytokines (IL-4, IL-10, and Alox15), chemokines (Ccl1 and Cxcr1), as well as the anti-inflammatory signature gene, TGF-β ( Fig. 5i and 5j)." (page 14-15, line 296-302) The following were added to the Discussion: "fourth, in the late phase of MI, the number of Trem2 hi macrophages abundantly expressing anti-inflammatory signature genes is significantly increased in the infarcted heart tissues and the soluble form of Trem2, the expression of which increased after the peak of fulllength Trem2 expression, significantly improves remodeling and cardiac function in the Regarding the cardiac function improvements in the GH-treated group without sTrem2, we have previously verified the effects of this gelatin-based hydrogel (GH) as already mentioned in the response to comment 11 (ACS Appl. Bio Mater. 2020, 3, 1646−1655. In our previous work, GH reduced the infarct size compared with the PBS-treated control, suggesting the potential impact of physical support to prevent infarct extension and LV dilation, which is consistent with the current results. Thus, GH hydrogels not only provide physical support for the infarcted myocardium but are also a suitable microenvironment for sTrem2 engraftment and its sustained release contributing to the cardiac regenerative effect. This statement has been added to the Discussion of the revised manuscript as follows: "Importantly, when we used an injectable gelatin-based hydrogel, which provided physical support for the infarcted myocardium and an appropriate microenvironment for simultaneously delivering biomolecules (ACS Appl. Bio Mater. 2020, 3, 1646−1655, the injection of sTrem2 with GH into the peri- Response: As the reviewer suggested, we have added the additional explanation to the Results section of the revised manuscript as follows: "The proportion of Ccr2 hi macrophages rapidly increased at day 1 post-MI and then declined over time (Fig. 5d), suggesting that monocytederived macrophages were recruited to the infarcted areas at early MI, which was consistent with a sub-clustering analysis of scRNA-seq data. In contrast, the proportion of Trem2 hi macrophages began to increase at day 3 and peaked 5 days after MI, indicating a specific dominance in late MI in the infarcted heart ( Fig. 5e-f)." (page 14, line 286-291) Comment 22. (Minor points) In material and methods, paragraph co-localization assay, the reference of the secondary antibodies is missing.

Response:
We have added the secondary antibody information (page 27, line 587-588).

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): With additional analyses, the manuscript has been improved somewhat. However, it still remains unclear whether Trem2+ macrophages are anti-inflammatory and sTrem2 is a key mediator produced by macrophages that control remodeling processes after MI. Additional analytical data now show that Trem2 is expressed in subsets of macrophages that express higher levels of Il10, Tgfb1, and Il4. Though authors note that they are anti-inflammatory macrophages, the expression profile suggests that they are also pro-fibrotic and pro-remodeling. Though ST-seq data are of interest, the mechanistic side of the study is very limited and provides insufficient evidence for the beneficial role of Trem2. Authors now abandoned M1/M2 dichotomy. However, they still rely on simple labeling of anti-inflammatory due to the expression of some signature genes, such as Arg1.
Expression of the genes suggests that Trem2-expressing macrophages contribute to ECM production/fibrosis in MI. This appears to be contradictory to the authors' claim that Trem2 ameliorates structural remodeling after MI. Previous studies have demonstrated that macrophages crucially contribute to scar formation. Though during scar formation and remodeling, they may also regulate inflammation, their potential function to lead remodeling should not be ignored.

Minor:
Gene names in graphs should be official gene symbols rather than conventional names (e.g., Fig.  5i, j).
Reviewer #2 (Remarks to the Author): Jung and colleagues have revised some of their statements that largely addresses my prior questions and concerns. The major issue was that ST-seq is not technically single-cell and this caveat needed to be discussed in the manuscript, which the authors have now done. The authors have also reanalyzed their data using SPOTlight in order to better infer the cell-type composition of each spot. While it does not fully address the caveats (and frankly the non-single-cell nature of the approach cannot be solved), it is a reasonable effort on their part. I would also note that the authors have performed new experiments and analyses to address the concerns. Thus, the clarifications and new data that they have provided have improved the manuscript, which I believe would be of interest to the field and broad readership of Nature Communications.
Reviewer #3 (Remarks to the Author): Single-cell transcriptomics unveils the spatiotemporal dynamics of macrophage heterogeneity and a potential role of Trem2hi macrophages in infarcted hearts The authors addressed my previous concerns nicely. However, I have a few additional comments. 3. Post-MI cardiac function is directly influenced by fibrosis, the indicator of stiffness of heart. While the effect of sTrem2 on improved cardiac function is well described, the contribution into global fibrosis is totally lack in this manuscript. Especially, reparative macrophages have been often shown to participate in fibrosis development, therefore, in addition to cardiac function, fibrosis measurement has to be added.
4. Authors' in vivo data looks really promising, especially there's big difference in survival rate between PBS and Trem2-GH. But sTrem2 is already high in d7 post-MI, is it not sufficient or not early enough? How's sTrem2 level in heart at d7 post-MI upon sTrem2 injection? In line with this, could you also provide PK study of sTrem2?

Response to reviewer comments
Reviewer #1 Comment 1. p. 14-15 "Ccr2 hi macrophages exhibited higher expression levels of proinflammatory cytokines (IL-1β and IL-6) and chemokines (Ccl2, Cxcr3, and Cxcr7), whereas Trem2hi macrophages expressed higher levels of anti-inflammatory cytokines (IL-4, IL-10, and Alox15), chemokines (Ccl1 and Cxcr1), as well as the anti-inflammatory signature gene, TGF-β ( Fig. 5i and 5j)." IL-4 is not an anti-inflammatory cytokine, and neither is CCL1. Cxcr1 is a receptor, and Alox15 is an enzyme. Please carefully revise these and other misleading and erroneous labeling and explanations of genes and cells.
Response: As suggested, we have removed the Il4 and Ccl1, and carefully corrected the misclassifications as follows. "In the quantitative reverse transcription-PCR analysis, Ccr2 hi macrophages exhibited higher expression levels of pro-inflammatory genes (cytokines: Il1b and Il6, chemokine: Ccl2, and chemokine receptors: Cxcr3 and Cxcr7), whereas Trem2 hi macrophages expressed higher levels of anti-inflammatory genes (cytokines: Il10 and Tgfb1, chemokine receptor: Cx3cr1, and enzyme: Alox15) ( Fig. 5i and 5j)." (page 14, line 293-298) Comment 2. Expression of the genes suggests that Trem2-expressing macrophages contribute to ECM production/fibrosis in MI. This appears to be contradictory to the authors' claim that Trem2 ameliorates structural remodeling after MI. Previous studies have demonstrated that macrophages crucially contribute to scar formation. Though during scar formation and remodeling, they may also regulate inflammation, their potential function to lead remodeling should not be ignored.

Response:
We appreciate the reviewer's valuable comment. It is well-known that cardiac repair after MI results from a finely orchestrated and complex series of events, initiated by an inflammatory phase (≈3-4 day in mice) with intense inflammatory responses, followed by a reparative phase (peak day ≈7 post-MI) with a resolution of inflammation, myofibroblast proliferation, and scar formation over the next several days, presenting increased expression of anti-inflammatory and profibrotic factors (eg, Il10 and Tgfb1). Therefore, the higher expression level of Il10 and Tgfb1 on days 5 and 7 post-MI seems reasonable. In addition, because it is not clear whether the increase in fibrosis-related gene expression leads to actual fibrosis development, we have further analyzed the wall thickness and collagen area in the infarcted heart tissues on day 28 post-MI. The MI mice treated with sTrem2 exhibited significantly thicker left ventricular wall and reduced collagen area in the infarcted region than the mice treated with PBS or GH alone, indicating that a local injection of sTrem2 induced a favorable LV remodeling after MI. The related statement has been added to the Result as follow: "Histologically, the mice treated with sTrem2-GH showed less dilated and well-remodeled LV with thicker infarcted walls and lower fibrosis ( Fig. 6b and Supplementary Fig. 16)." (page 15, line 320-322) <Supplementary figure 16; n=4-7, * P<0.05, ns: not significant> Comment 3. (Minor points) Gene names in graphs should be official gene symbols rather than conventional names (e.g., Fig. 5i, j). This can be simply checked by secreted level of sTrem2 from Trem2 hi macrophages or Trem2 overexpression macrophages in vitro.

Response:
As the reviewer suggested, we have evaluated the secretion levels of sTrem2 in Trem2 hi and Trem2 low macrophages in this revision. For this, F4/80 + Trem2 hi and F4/80 + Trem2 low macrophage subsets were sorted from the infarcted heart tissue on days 3 (Trem2 hi macrophages began to increase) and 5 (Trem2 hi macrophages reached a peak) post-MI. The sorted cells were cultured for 2 hours and the secretion levels of sTrem2 was measured by ELISA. When we compared the secretion levels of sTrem2 between Trem2 hi and Trem2 low macrophages, sTrem2 was detected only in Trem2 hi macrophages (see below figure). Of note, the secretion level of sTrem2 on day 5 was significantly higher than that on day 3, which was consistent with western blotting analysis. This data has been added to a supplementary figure with relevant statements in the Result section as follows: "We also evaluated the secretion levels of sTrem2 in Trem2 hi and Trem2 low macrophages from days 3 and 5 post-MI and identified that sTrem2 was secreted only in Trem2 hi macrophages ( Supplementary Fig. 12). Of note, the secretion level of sTrem2 on day 5 was significantly higher than that on day 3.
Collectively, these results indicate that Trem2 is highly expressed by macrophages in the infarcted heart and sTrem2 is exclusively secreted from Trem2 hi macrophages." (page 13-14, line 271-276) <Supplementary figure 12; n=6-8, ** P<0.01, n.d.: not detected> Comment 3. Post-MI cardiac function is directly influenced by fibrosis, the indicator of stiffness of heart. While the effect of sTrem2 on improved cardiac function is well described, the contribution into global fibrosis is totally lack in this manuscript. Especially, reparative macrophages have been often shown to participate in fibrosis development, therefore, in addition to cardiac function, fibrosis measurement has to be added.

Response:
We appreciate this valuable comment. To address this issue, we have further analyzed the wall thickness and collagen area in the infarcted heart tissues on day 28 post-MI.
The MI mice treated with sTrem2 exhibited significantly thicker left ventricular wall and reduced collagen area in the infarcted region than the mice treated with PBS or GH alone,